Gene regulation plays key roles in many aspects of disease and development. The human genome project and other sequencing efforts have provided much information on the identity of genes. Functional studies have examined how genes affect physiological and pathological processes. Living organisms use various mechanisms to regulate gene expression and thereby affect the onset and progression of many diseases as well as multiple processes observed under non-disease conditions. The ability to modulate gene expression would provide a means of adjusting physiological processes in disease and non-disease conditions, especially where the modulation of gene expression can be aimed at select genes of interest. Of particular interest are compounds capable of modulating gene expression in cultured cells, in animals, in plants, and/or in humans.
Among compounds capable of modulating gene expression are polyamides, which have been used for that purpose in cells and animals. Polyamides have been shown to bind DNA at specific sequences, thus allowing them to modulate a gene linked to those sequences while not or not significantly interfering with the expression of other genes. Moreover, polyamides can be designed to recognize a DNA sequence of interest, thus allowing drug design aimed at modulating the expression of genes involved in a disease or phenotype of interest. While the sequence-specific DNA binding ability of polyamides has been recognized, it has been difficult to facilitate delivery of polyamides to their target sequence, while maintaining their DNA binding abilities. Target delivery of polyamides is believed to involve uptake of the polyamides into cells and their nuclei, and it is further aided by their ability to sufficiently remain in the nuclei to allow binding of the polyamides to their target sequence.
The entry of molecules into cells and their nuclei is generally believed to inversely correlate with the size of the molecules. Polyamides are large and complex molecules when compared to typical molecules known to enter cells and their nuclei with ease. Polyamides were therefore traditionally designed to minimize their size to facilitate entry into cells and their nuclei. Polyamides that were limited to structures needed for DNA binding, however, were found to have limited abilities to enter cells and especially their nuclei. Polyamides linked to the large fluorophore fluorescein, by contrast, were found to have significantly enhanced capabilities to enter cells and nuclei. This finding seemed surprising given that the fluorescein molecule carries two negative charges under physiological conditions and includes four six-rings, thus considerably increasing the size of the polyamide.
The addition of fluorescein to polyamides, however, has certain disadvantages. For example, the fluorescein moiety is not considered necessary for DNA binding but rather was found to lessen the affinity of polyamides for their target DNA. Also, the conversion of polyamides to polyamide-fluorescein conjugates requires additional synthesis steps and thus increases the cost of polyamides and their use.
Thus, new polyamide derivates are needed that have a high DNA binding affinity and that are capable of entering cells and their nuclei. The current invention provides such polyamides.